2369
Tautomerism of Purines. I. N(7)H Equilibrium in Adenine'
N(9)H
M. Dreyfus, G . Dodin, 0. Bensaude, and J. E. Dubois* Contribution from the Laboratoire de Chimie Organique Physique, associs au C.N.R.S., Llniversit5 Paris VU, 75005, Paris, France. Received June 8, 1974
Abstract: Neutral adenine in water exists in two tautomeric forms, N(7)H and N(9)H adenine. The equilibrium constant, K = CN(7)H/CN(P)H, is estimated to be 0.28 at 20°, and the enthalpy of tautomerization is 0.2 kcal/mol. The interconversion between the two forms is catalyzed by H+ ( k ~ =+ 1.3 X 1O1OM-' sec-' at lo'), by OH- OH- = 0.85 X 1Olo M-' sec-I), and by the anion of adenine ( k ~ =- 3.5 X lo8 M-l sec-l). Due to the low proportion of the N(7)HN(9)H+ cation in protonated adenine, no acidic autocatalysis is observed.
Numerous works both theoretical2 and experimental3 have been devoted to the tautomerism of purine bases. The major importance of this question is linked to the fact that the reactivity of the biological purines will depend on the structure and relative concentration of the various species present in aqueous solutions. Formal structures of the purine molecule as well as theoretical considerations show the existence of several tautomeric forms arising either (a) from prc.totropy involving an extranuclear atom (e.g., ketoenol and amine-imine tautomerism), or (b) prototropy involving only ring atoms (e.g., the N(l)H, N(3)H, N(7)H, and N(9)H tautomers of purine). Uv4 and irs spectra of oxo- and aminopurines and their methylated derivatives do not provide any conclusive evidence of amine-imine or keto-enol equilibria and, conselquently, suggest that these purines exist mainly in the keto and amino forms (with the exception of 1-methyladenine which consists mainly of the imino tautomer6). On the other hand, tautomerism involving ring atoms only has been clearly observed. To account for the ir spectrum of hypoxanthine Brown and Mason5 postulated an equilibrium between the N('i)H and N(9)H forms. Pfleiderer7 proposed that the uv spectrum of guanine in water must be interpreted in terms of a contribution from N(7)H and N (9) H tautomers. More recently, quantitative studies have led to estimaN ( 9 ) H equilibrium; tions of the constant for the N(7)H CI3 nmr data from concentrated aqueous solutions of purine suggest that both tautomers are present in equal proportions.s Fluorescence experiments conclude that adenine in 2-butanol at 170'K consists of 6% N(7)H and 94% N ( 9 ) H forms.9 Two striking conclusions may be drawn from the examination of the abundant literature: (a) there are few thermodynamic data for the observed tautomeric equilibria N(7)H F! N(9)H for biological purines, (b) quantitative kinetic investigations of the interconversion are totally lacking. These facts prompted us to approach the question of tautomerism by means of a fast kinetic method. W e would now like to report an investigation of N(7)H e N(9)H tautomerism of adenine in water using the T-jump relaxation technique.'O h", NH:, I
I
N(9)H N(7)H Experimental Section Adenine (Biochemicals) and 9-methyladenine (Cyclochemicals) were recrystallized three times from water; 7-methyladenine was
prepared according to published and purified by successive crystallization from water and absolute ethanol. NaC104 H20 (Merck reagent grade) was used without further purification. Solutions for the kinetic runs were prepared by dissolving weighted amounts of bases in 0.2 M solutions of NaC104 in distilled water. The solutions were degassed prior to use. T-Jump Apparatus. Temperature-jump measurements were performed using a Joule-heated double beam spectrometer (Messanlagen Studiengesellschaft). A special sample cell was designed so as to allow the solution under investigation to circulate by means of a peristaltic pump. Before entering the cell the solution stands in a thermostated glass reservoir whose volume is much larger than that of the sample cell (30 cm3 vs. 0.4 cm3). This system ensures rapid homogenization of both pH and temperature throughout the sample solution. The reservoir was designed to permit the immersion in the circulating liquid of a thermometer (temperature ti) and two Beckman electrodes (G.P. glass electrode No. 39000 and calomel reference electrode No. 39402). The whole system is protected from atmospheric contact; a septum is fitted on the reservoir thus permitting the pH to be varied by injecting small amounts of NaOH or HCI. All connections are made using 2 mm i.d. PTFE or VITON (in the peristaltic pump) tubing. Filling of the system previously purged with argon was made by pumping the solution directly from the flask used for evacuation, thereby allowing minimum exposure to air. The flow rate on the system is about 80 cm3/min during the experiments. Blank runs were performed with a thermistance inserted into the T-jump cell and it was checked that the temperature difference between the reservoir and the sample cell is negligible using this flow rate. The replacement of the T-jumped liquid was slow enough not to interfere with the relaxation times which are observed (T < 600 fisec) and consequently the pumping of the sohtions was not stopped during the temperature jump. The initial temperature ( t i ) was controlled within 10.2O by a Lauda U.K. 20 cryostat in the temperature range 0-20' and a Colora thermostat from 20' to 80". The temperature following the jump ( t f ) was computed from t i and the temperature jump magnitude, which was estimated as follows: the circulating system was filled with a solution showing a relaxation phenomenon of large amplitude; solutions of 3-hydroxypyridine, which exhibits tautomerism, were found convenient for this purpose. The relaxation amplitude for a given discharging voltage was recorded, and then ti was rised with the aid of the thermostat so as to produce the same oscilloscope deflection as the relaxation experiment; the corresponding change of ti was taken as the T-jump magnitude: it was 9 1 0.5' in all experiments reported here. Jumps of such magnitude were found feasible in the range 0-80° (ti) when carefully evacuated solutions were used. However, cavitation phenomena cause increasing troubles at the high temperatures. pH Measuremnts. The pH was measured with a Beckman 1019 research pH meter; during the temperature jump the electrodes were disconnected from the pH meter to avoid damaging its high impedance preamplifier. It was found difficult to obtain reliable pH measurements in cold NaC104 solutions with a conventional reference electrode, due to partial clogging of fritted junctions by slightly soluble KC104. This was finally avoided by replacing satu-
-
Dubois et ai.
/
N(7)H$N(9)H Equilibrium in Adenine
2370
Figure 1. T-Jump rclaxation spectrum of adenine in aqueous solution recorded far a wavelength of 280 nm: initial temperature (11) = lo:, final temperature (it) = I O o ; pH (as measured at ti) = 7.W.adenine O.D. unit per concentration = 9.5 X I O P M. Vertical scale: 5 X major division. Horizontal scale: 200 p e c per major division. The rignal corresponds to an increase of the solution O.D.
rated KCI solution by saturated NaCl solution in the calomel electrode. Such a modified electrode gives stable measurements, but there is evidence that its liquid junction potential is not independent of the solution in which it is immersed; thus, the emf of the cell HA [Hg2CI2]KCl(sat)lsolution XINaCl(sat)[HgzCIz]1Hg was found to vary significantly when changing the buffer solution X. In order to estimate the systematic error thus introduced in pH measurements. the following procedure was used: after the pH meter had been standardized in the usual way with Beckman precision phosphate buffer (part No. 566008). we measured the pK of several indicators in 0.2 M NaC104 solutions: the circulating system was employed for this purpose, the T-jump detection being used as a spectrophotometer to record the variation of optical transmission when changing pH. The following values were found, at l o (after activity correction has been made): 0-nitrophenol 7.65 (lit." 7.52); p-nitrophenol 7.55 (lit." 7.43); 3-hydroxypyridine 5.1 I (lit." 5.05). This suggests that the measured pH should be corrected by about 0.1 pH unit to yield accurate values. pH measurements quoted in the text have been corrected in that way. Although the capacitors were discharged up to a hundred time in a typical kinetic run, no alteration of the solutions is observed: this is demonstrated by the high reproducibility of the measured relaxation times. Data Processing. The voltage output from the T-jump spectrometer is fed into a PDP-I I processor (Digital Equipment Corp.). The structure of this wmputer makes it specially wnvenient for high speed direct access to wre memory. A special interface was built in our laboratory for this purpose; only a brief description of this device will be given here, as a more detailed report will appear elsewhere.'5 The signal from the T-jump is first amplified, then wnverted by a high speed IO bits A/D wnverter (DATEL); the sampling rate, and the number of values to be transmitted in wre memory as well as the delay between the triggering of the capacitors and the beginning of the acquisition process may be selected: the maximum sampling rate is 2 wsec per value. Delayed acquisition is used to eliminate very fast relaxation phenomena not to be studied. The signal to noise ratio is poor (S/N = 2 to 3) at the lowest concentrations of adenine we have used, therefore, it was found valuable to accumulate 3 to 6 relaxation curves, thereby increasing S/N by a factor of about 2. The signal thus obtained is smoothed by a procedure described by Savitzky et a1.I6 and then translated so that the average of the last 20 values is put equal to zero;finally a least-square procedure is used to calculate the relaxation time by means of eq I .
h y =
-UT
+hyn
(1)
A more sophisticated alternative is to search for an optimum base line by incrementing all the y's in eq I so that a maximum for the correlatton coefficient is reached: this procedure was not.found to increase significantly the reproducibility of the measurements. When the best fit is obtained for T, the amplitude yo, extrapolated to zero delay, is computed. From the gain of the interface, the amplitude ( A ) of the relaxation expressed in optical density units is then readily obtained." For each pH value at least five series of three to six curves were processed. The standard deviation is used as an estimate of the un-
I U
-5m
I 2%(
mn
I
270
I
281
I 190
I
IC0
Llnml Figure 2. (a) Ratio of the relaxation amplitude to the adenine concentration versus the wavelength li = lo; I ( = IO': uncertainties are the standard deviations. (b) Plat of the molar extinction coefficientdifference ( C N ( I ) M ~- . M X io4 DHQ x 10-4 x 10-4 r-3.13 6.34 1.0 (0.05)b 1.02 3.13 0.324 (0.004) 0.348 6.86 3.13 7.83 0.24 0.22 (0.03) 0.49 3.13 8.26 0.52 (0.03) 0.75 3.13 8.46 0.78 (0.04) 7.23 5.78 3.53 3.57 (0.34) 7.23 1.04 6.33 1.01 (0.01) 0.72 7.23 6.50 0.70 (0.02) 7.23 0.48 6.69 0.453 (0.02) 0.42 7.23 6.77 0.39 (0.04) 7.23 7.40 0.211 0.202 (0.002) 7.23 7.95 0.34 (0.02) 0.344 7.23 0.67 8.30 0.72 (0.04) 7.23 1.01 8.49 1.03 (0.05) 7.23 8.57 1.19 1.18 (0.04) 7.23 2.44 8.89 2.26 (0.03) 9.45 6.06 1.95 (0.13) 1.91 9.45 6.34 1.02 1.13 (0.07) 9.45 0.82 6.44 0.91 (0.03) 9.45 6.64 0.54 0.59 (0.01) 9.45 7.78 0.296 0.298 (0.002) 9.45 0.381 7.95 0.376 (0.017) 9.45 8.17 0.605 0.59 (0.02) 9.45 0.86 8.37 0.97 (0.02) 9.45 1.28 8.55 1.40 (0.04) 1.46 6.18 1.33 (0.02) 11.5 0.294 7.00 11.5 0.283 (0.003) 11.5 0.533 8.09 0.508 (0.004) 1.07 8.44 1.09 (0.02) 11.5 1.48 8.58 1.46 (0.04) 11.5 4.79 9.11 4.72 (0.10) 11.5 a As measured at ti. b Standard deviation. C Computed from eq 2 (see text). \
Figure 3. Ratio of the final-to-initial equilibrium concentrations of various species in adenine solution as a function of pH: ti = l o ; tf = loo; concentration of adenine = 4.18 X M. (a) OH-, (b) H+, (c) anionic adenine, (d) cationic adenine.
L
7
6
D
-log
CH+
Figure 4. Log T-' as a function of -log E"+. Adenine concentration = 7.23 X M ; t i = 1'; tf = IOo;X 280 nm.
I
The amplitude, measured in the p H range where it is p H independent, was found to decrease rapidly when the temperature was raised [Table I) and becomes vanishingly small a t temperatures above 50'. Relaxation Time. For t i = 1' and tf = lo', the relaxation time was measured over more than 3 pH units and was found to be very sensitive to the pH (Table 11). The relaxation times do not show any systematic dependence upon the adenine concentration for pH < 7.3 but at higher pH values definite evidence for a decrease of 7-l was observed when adenine concentration was decreased. The rather large enthalpy of dissociation of water causes the equilibrium concentrations of hydroxide and hydronium ions to differ considerably a t ti and a t tf when the temperature jump is-9'. For reasons to be discussed below, the concentrations CH+and COH-corresponding to the final temperature should be considered in the interpretation of the kinetic results rather than EH+' and &-'whigh refer to t_he initial temperature. The concentrations CH+' and COH-', as well as the concentrations of adenine in cationic, neutral, and anionic forms before the jump, were computed18 from the measured p H and the dissociation constants
of the cationic (K1) and neutral ( K z ) forms of adenine a t the corresponding temperature evaluated from the literature data.19 These concentrations were then used to compute the equilibrium concentrations a t t f according to the procedure described in the Appendix. A plot of C H + / C H + O and &H-/COH-O as a function of p H (Figure 3) for a typical adenine concentration shows that these ratios deviate considerably from unity. The variation of 7 - l with the pH and the concentration of adenine was fitted by the equation A least-squares fitting leads to ko = 210 sec-', k = 1.3 X 1Olo M-' sec-I, k' = 0.85 X 1Olo M-' sec-', and k" = 6.5 X lo1*M-I sec-'. Although probably real, the term ko contributes little to the rate law even a t neutral p H where 7-l is minimum, so that a plot of log 7-l versus log CH+for a fixed adenine concentration shows two straight lines with unit slope, connected by a valley (Figure 4).
Discussion The amplitude ( A ) of the relaxation process for a twospecies equilibrium as monitored by uv spectroscopy is readily derived A = C[ZK/(l + K ) ' ] A < A T A H 0 / R T 2 (3) Dubois et al.
/
N(7)H=N(9)H Equilibrium in Adenine
2312
t-
1
1.5
t
0.50
0.25 _ I I
~
0
,
I
1-1-1
280
275
' *
1-1
I
,!(nm
-
Figure 5. r = ((adenine' (N(9)MeX+2'5)/((N(7)Me'+2'5 as a function of X (see text).
285
- W(9)Me'+2'5)
where C is the total concentration, I is the optical length, AT is the magnitude of the temperature jump, A€ is the difference between the molar extinction coefficients of each species, A H 0 is the reaction enthalpy, and K is the equilibrium constant. Thus, for such a process, the amplitude-to-concentration ratio for a given temperature and A T is proportional to the difference of the molar extinction coefficients of the two interconverting species. The close similarity of the wavelength dependence of the amplitude-to-concentration ratio (Figure 2a) with the differential spectrum of 7-methyladenine and 9-methyladenine (Figure 2b) demonstrates that the observed relaxation is as-. sociated with the reequilibrium of the N ( 7 ) H and N ( 9 ) H tautomers of adenine in water. The p H independence of the amplitude and its proportionality to adenine concentration are consistent with this interpretation.
Estimate of the Thermodynamic Parameters of the N(7)H N(9)H Equilibrium. The overall molar extinction coefficient for adenine is €adenine
= (1 -
01)EN(9)H
'
=
(€adenine
-
EN(S)H)/(EN(7)H
O1> k23; this means that the acid BH+ experiences many ionizations followed by back capture of the proton, before finally dissociation takes place. Now, when B has several basic sites, it is suggested that the proton ionizing from one basic center can occasionally be trapped back on another, and thereby that interconversion of the tautomeric species occurs efficiently without acid dissociation; the adeninium cation may then exist under all its possible tautomeric forms during its lifetime. If this “proton scrambling” mechanism is operative in the adeninium cation, k4 and k-5 then must represent the rate constant for overall protonation of N ( 9 ) H and N(7)H adenine, rather than for protonation on the sole imidazole ring, +~” and, indeed, the value found for ( k d k - - ~ ) / k ~compares favorably with the rate constant for protonation of imidazole38,39and possibly for overall protonation of purine.32 In addition to Scheme 111, several reactions schemes for the acid-catalyzed N(7)H + N(9)H interconversion may formally be written if proton scrambling occurs; for example, see Scheme IV. Scheme IV “2
I
XH,
NH2
the interconversion of the various adeninium cations, contributes little to the N ( 7 ) H e N ( 9 ) H exchange in adenine. However, the respective spatial positions of the proton donor and proton acceptor groups in adenine, pointing in opposite directions, clearly do not favor such a mechanism; it may nevertheless hold for a more favorable geometry, and thus our failure to detect the amine-imine equilibrium in adenine or methylated analogs cannot be considered as definite evidence against the existence of such tautomerism (although it seems unlikely both on a theoretical and an experimental b a s i ~ ) . In ~ . ~that case, N ( l ) and N(10) would be linked by a chain of H bonds involving only one or two water molecules and thus a proton exchange between these two sites is likely to proceed via a very fast concerted mechanism rather than a acid- and base-catalyzed one.
Acknowledgment. We are much indebted to J. C . Fontaine, J. J. Meyer, and P. Levoir for technical help with the electronics and the programming of the data processing system. Appendix Computation of the Relaxation Times and Amplitudes for a Given Mechanism. A general formalism allowing the computation of the relaxation spectrum associated with a reaction mechanism of any complexity has been derived by Cast e l l a r ~ .This ~ ~ treatment may also be used to evaluate the respective amplitudes, expressed as concentration changes, associated with the successive relaxation times. The notations are from ref 41. Consider a reaction mechanism involving N chemical species and consisting of R elementary chemical reactions, among which R’ are linearly independent. The stoichiometric coefficient, via of the chemical species i in the reaction a will be taken as negative for the reactants and positive for the products. When the system is slightly displaced from equilibrium, this mechanism is characterized by a relaxation spectrum. The inverses of the relaxation times may be defined as the solutions of the eigenvalue problem
BU = UX Such types of mechanism have been proposed to explain the kinetics of proton exchange in acidic solutions of puHowever, tautomers of adenine lacking hydrogen on the imidazole ring are likely to be very unstable compared to the N ( 7 ) H or N(9)H tautomer,26 so that Scheme IV is probably a rather high energy process. It seems more probable that the imidazole ring always retains a proton during reversible ionization of the adeninium cation, thereby making the N(7)HN(9)H+ species a necessary intermediate in the N(9)H 8 N(7)H acid-catalyzed interconversion.
(1)
where B is a symmetrical matrix (several other forms of this eigenvalue problem are given in ref 41; however, diagonalization of a symmetrical matrix has distincts advantages for machine computation) expressed as
B
r is a
R
X
=
.1/2G,1/2
( 2) R diagonal square matrix, the nonzero elements
of which are the “exchange currents” associated with the corresponding chemical reaction; G is a R X R symmetrical matrix defined by
Conclusion The temperature-jump technique has proved successful in investigating tautomerism in aqueous adenine both in neutral and protonated forms; our preliminary observations on guanine solutions show that it can be usefully applied to other purines as well. The rate of interconversion of the two tautomers of neutral adenine was found to be catalyzed by ,acids and bases; in the neutral pH range where the rate is the slowest, the equilibrium process is virtually completed within 1 msec a t 10’. The proposed mechanism for this interconversion involving the exchange of a proton between two nitrogen atoms in water is quite similar to the wellknown mechanism of the keto-enol interconversion of carbonyl compounds40 although the rate constants involved here are considerably larger. The pH-independent term in the rate law is very small; this shows that the intramolecular, water-catalyzed mechanism, which probably holds for Journal of the American Chemical Society
/ 97:9 /
where Ci is the molar concentration of the chemical species i. The difference between the “reaction coordinate” of the reaction a at a given time and its final equilibrium value may be computed from the eigenvalues and eigenvectors of
B
u,,
Ata =
Ace%‘
(3)
E
where y , = A , e - X J is the “normal coordinate of relaxation” corresponding to the relaxation time 7, = l/k,. (Equation 3 is equivalent to eq 70 of ref 41 .) If some elementary reactions can be expressed as linear combinations of others, viz., if R’ < R then B has only R’ nonzero eigenvalues. To the remaining ( R - R’) zero roots correspond zero amplitude normal coordinates (cf. Castel-
April 30, 1975
2315 lan's eq 83) so that only the R' nonzero modes need to be considered in the summation over E in eq 3. The difference between the concentration Ci of the species i at a given time and its final equilibrium value may be computed by
E.1
[Y
Thus, the overall concentration change of i during the relaxation mode e is proportional to the coefficient bi, = C[YUol,vi[Yra1/2. The comparison of the different bi, for a given relaxation mode e gives information on the "composition" of this mode in terms of concentration changes. Now, the determination of the absolute concentration changes accompanying the successive relaxation modes necessitates the determination of their respective amplitudes, A,. If the concentrations corresponding to the initial equilibrium Ci0 together with the final ones are known, the A , may be obtained by solving the linear system
(5)
(5) is a set of N equations, R' of them being independent, of the R' unknowns A , . A general program was written in Fortran to build the symmetric matrix B from the final concentrations ci, computed as below, and approximate values of the rate constants. Rate constants of 1Olo M-' sec-' were used for all the elementary exothermic reactions,30 except those involving two different forms of the adenine molecule; a value of lo9 M-1 sec-' was chosen in that case to account for the smaller diffusion coefficients of large molecules compared to those of H+ and OH-. B was then diagonalized using the Givens procedure to obtain the relaxation spectrum; the "composition" of each successive relaxation was investigated by computing the coefficients bi, from the eigenvectors of B. The absolute amplitude of each normal mode was then obtained by solving the system 5. Computation of the Concentrations Corresponding to the Final Equilibrium. It will be assumed that a set of R' independent chemical reactions has been chosen; the equilibrium constant of the independent reaction X will be denoted Ria and at the initial and final temperatures, respectiveof the species i can be writly. The final concentration, ten as
ci,
A=i
where the
must satisfy the following system
.%A
References and Notes (1) This article will be included in the doctoral thesis of M. Dreyfus, Universite de Paris VII. (2) (a) B. Pullman, "Jerusalem Symposia on Quantum Chemistry and Biochemistry", Vol. II, E. D. Bergmann and 8. Pullman, Ed., Jerusalem, 1970, and references therein: (b) 8. and A. Pullman, "Advances in Heterocyclic Chemistry", Vol. 13, Academic Press, New York, N.Y., 1971. (3) (a) P. D. Lawley. "Fused Pyrimidines", Part 11, "Purines", D. J. Brown, Ed., Wiiey-lnterscience, New York, N.Y., 1971, p 439. (b) R. Lumley Jones, ibid., p 496. (4) S. F. Mason, "Chemistry and Biology of Purines", Wolstenholme and O'Connor, Ed., J. and A. Churchill Ltd.. London, 1957, and references therein. (5) D. J. Brown and S. F. Mason, J. Chem. SOC.,682 (1957). (6) P. Brookes and P. D. Lawley, J . Chem. SOC., 539 (1960). (7) W. Pfleiderer, Justus Liebigs Ann. Chem., 647, 167 (1961). (8) R. J. Pugmire and D. M. Grant, J. Am. Chem. Soc., 93, 1880 (1971). (9) J. W. Eastman, Ber. Bunsenges. Phys. Chem., 73,407 (1969). (10) M. Eigen and L. De Maeyer, "Technique of Organic Chemistry", Vol. VII. Part II, S. L. Friess, E. S.Lewis, and A. Weissberger, Ed., Interscience, New York, N.Y., 1963. (1 1) N. J. Leonard and T. Fujii, J. Am. Chem. SOC.,85, 3719 (1963). (12) 2. Neiman and F. Bergmann, Isr. J. Chem., 6, 9 (1968). (13) L. P. Fernandez and L. G. Hepler, J. Am. Chem. SOC.,81, 1783 (1959). (14) J. J. Christensen, D. E. Smith, M. D. Slade, and R. M. Izatt, Thermochim. Acta. ~. 5. 35 119721. > -, (15) J.-A. Miller, P. Levoir. J. C. Fontaine, F. Garnier, and J. E. Dubois, Anal. Chem.. 47. 29 11975). (16) A. Savitzky and M. J.'E. Golay, Anal. Chem., 36, 1627 (1964). (17) The detection system of the T-jump spectrometer is factory adjusted so that a variation of the output voltage by 10 mV corresponds to a change of the optical density of 0.001 unit. (18) The Debye-Huckel formula log f = - O . S l f i ( l 4- 0.3adO was used to estimate the activity coefficients of monovalent ions in 0.2 M NaClO, solutions: a value of 3 A is chosen for all ions except H+ (a = 9 A). (19) J. J. Christensen, J. H. Rytting, and R. M. Izatt, Biochemistry, 9, 4907 (1970). (20) Some furthor justification for this hypothesis can be gained from our knowledge of the spectral properties of purines. A large amount of work has been devote&' to this subject since the early work of Mason:22although the available experimental and theoretical evidences are sometimes conflicting, particularly as concerns the polarization direction of the different electronic transitionsF3 the following pattern emerges: the long-wavelength electronic band of adenine is in fact a composite one, its two components having different intensities: the strongest and weakest are referred to as x j and x2, respectively, by Mason.22 In related compounds such as benzimidazoles, X I and x2 appear as distinct bands, x 1 being the long-wavelength one.22 The effect of methylation on the spectra of these nontautomeric systems can be studied; it is found that the x1 band is red shifted upon methylation by about 3 nm and the x2 band somewhat It is obvious from Figure 6 that the spectrum of adenine and the weighted spectrum of 22% 7-methyladenine and 78% 9-methyladenine are similar both in shape and intensity with a shift of 2.5 to 3 nm except in the spectral range 262-274 nm where the shift is apparently larger: this may correspond to the weak x 2 band. (21) W. Hug and I. Tinoco, Jr.. J. Am. Chem. SOC.,95, 2803 (1973), and references therein. (22) S. F. Mason, J. Chem. SOC.,2071 (1954). (23) R . F. Stewart and N. Davidson, J. Chem. Phys., 39, 255 (1963). (24) G. H. Beaven, E. R . Holiday, and E. A. Johnson, Spectrochim. Acta, 4, 338 (1951). (25) F. Jordan and H. D. Sostman, J . Am. Chem. SOC.,94, 7898 (1972). (26) B. Pullman, H. Berthod, and M. Dreyfus. Theor. Chim. Acta, 15, 265 (1969). (27) E. D. Bergman, H. Weiler-Feilchenfeld. and Z. Neiman, J. Chem. SOC.B, 1334 (1970). (28) S. F. Mason, J. Chem. SOC.,5010 (1957). (29) If N(7)HN(9)H+ is assumed to be the onlv form of Drotonated adenine. six chemical species, namely the four fohns of adenine, H+, and OH-; are present: 14 chemical reactions relating them can be written: their equilibrium constants can be computed from the pKs of adenine,lD Kw, and our estimate for K. Four relaxation times are computed for this system: only the slowest has the observed order of magnitude and dependence on pH. It is found that: (a) only the reactions of Schemes I, 11, and 111, together with eq i N(9)H N(7)HN(9)H+ 2 N(7)H N(7)HN(9)H+ make an appreciable contribution to this relaxation mode; (b) that the concentration changes of N(7)H and N(9)H during this mode have opposite signs and a nearly equal magnitude: they vary little with pH _even - CN~~M') and (CN(,~ though the overall concentration changes (CN(S)H CN(~Y')vary much more; (c) the relative concentration changes of the various species during this process is very small: thus, despite the fact that the overall relative concentration changes may be large, cf. Figure 3, the use of a formalism valid only for small concentration changes appears justified: and (d) the relaxation time can be computed, to a good approximation, by assuming that A- and N(7)HN(9)H+ are in a steady state and consequently the relaxation mode can be regarded as a first-order process. Although accounting for most of the observations, this model erroneously predicts a strong acid autocatalysis via eq i; this is discussed below. M. Eigen, Angew. Chem., Int. Ed. Engl., 3, 1 (1964). H. G. Busse and G. Maass, 2. Phys. Chem. (Frankfurl am Main), 66, 92 (1969). T. H. Marshall and E. Grunwald. J. Am. Chem. SOC.,91, 4541 (1969).
~.
~~
~
+
(p = l...R')
(7)
(7) is a system of R' nonlinear equations of the R' unknowns XX. It may be solved for the by minimizing the following expression F =
R'
[E,
u =l
R'
i=l
]""]' ( 8 )
with respect to the xi; F should be zero for x i = Xi. A general routine of minimization of a several-variable function was used for this purpose, starting from X A = 0 as trial values for the .%A. The values of the Ci were finally obtained from the computed Xi by eq 6.
+
-
Dubois et al.
/
N ( 7 J H S N ( 9 ) HEquilibrium in Adenine
2376 (33) H. C. Borresen, Acta Chem. Scand., 21, 2463 (1967). (34) W. C. Coburn, Jr., M. C. Thorpe, J. A. Montgomery, and K. Hewson, J. Org. Chem.,30, 1114(1965). (35) R. Bonaccorsi, A. Pullman, E. Scrocco, and J. Tomasi, Theor. Chim. Acta, 24, 5 1 (1972). (36) E. Grunwald. "Progress in Physical Organic Chemistry", Vol. 3, Intersclence, New York, N.Y., 1965. (37) A value of 5.2 X 10" M' sec-' is found if the four basic sites are assumed to have identical reactivities. This value can be replaced in Debye formula for a diffusion-controlled reaction between a neutral and Dadenine),where u is the reaction a charged specles: k = 45?NPu(&+
+
distance, P is the steric factor, the diffusion coefficient &+ was computed from the mobility of the hydronium ion at 10'. and Dadnine was taken from W. J. Bowen and H. L. Martin, Arch. Siochem. Biophys., 107, 30 (1964) This leads to Pu = 9 A, a value far larger than those observed for simple amines.36 (38) E. K. Ralph and E. Grunwald, J. Am. Chem. SOC.,91, 2422 (1969). (39) M. Elgen, G. G. Hammes, and K. Kustin, J. Am. Chem. Soc., 82, 3482
(1960). (40) R . P. Bell, "The Proton in Chemistry", Methuen and Co. Ltd., London 1959. (41) G. W. Castellan. Ber. Bunsenges. Phys. Chem., 67, 898 (1963)
Interaction of Metal Ions with 8-Azapurines. Synthesis and Structure of Tetrachlorobis-2[( 5-amino-4-carboxamidinium)[ 1,2,3]triazole]copper(11) Monohydrate Linda Goodrich Purnell, Julianna Causey Shepherd,' and Derek J. Hodgson* Contribution from the Department of Chemistry. The University of North Carolina, Chapel Hill, North Carolina 27514. Received November 7 , 1974
Abstract: The reaction of copper(I1) chloride with 8-azaadenine in 0.36 M hydrochloric acid solution brings about ring opening at C(2) of the azapurine to form tetrachlorobis-2-[ (5-amino-4-carboxamidinium)[ 1,2,3]triazole]copper(II) monohyto as [CUCI~(HACT)~].H~O. The composition and geometry of this drate, [ C U C ~ ~ ( N ~ C ~ H ~ hereinafter ) ~ ] . H ~ Oreferred ,
complex have been verified by single-crystal X-ray crystallography using data collected by counter methods on an a'utomatic diffractometer. The material crystallizes in the monoclinic space group C2/c with four molecules in a cell of dimensions a = 15.153 (14) A, b = 6.962 (4) A, c = 18.274 (16) A, and /3 = 121.47 (6)O. The observed and calculated densities are 1.90 (3) and 1.93 g ~ m - respectively. ~, Least-squares refinement of the structure has led to a final value of the conventional R factor (on F) of 0.042 using 1131 independent intensities. The complex consists of monomeric CuC14 (HACT)2 units which are hydrogen bonded to the water molecules. The geometry at the copper(I1) centers is tetragonally distorted octahedral, the base plane being formed by two trans chlorine atoms and two trans N(8) atoms of the HACT+ ligands, while the axial ligands are the remaining chlorides; the copper atoms lie on a crystallographic inversion center. The in-plane Cu-N and Cu-CI distances of 2.049 (3) and 2.258 (2) A, respectively, are normal, as is the axial Cu-CI separation of 2.967 (2) A. The triazole portion of the ligand is planar, but the copper atom and the N and C atoms of the arnidine moiety are out of this plane. The ligand bond lengths suggest four principal resonance contributors to the bonding in HACT+. The hydrogen bonding in the crystals is complex, with all available donor and acceptor atoms apparently participating. Since neither adenine nor 8-azaguanine undergoes hydrolysis in weakly acidic solution, it appears probable that copper coordination at N(8) may assist ring opening at C(2) in 8-azapurines
The interactions of metal ions with nucleic acid constituents have been the subjects of numerous recent chemical, spectroscopic, and crystallographic studies;2-16 this demonstrated interest in this field is due primarily to the biological significance of metal-nucleic acid and peptide-metal-nucleic acid interactions in living system^.^^^^^-^^ Crystallographic studies have demonstrated that individual transition metal ions can bind in a variety of different ways to a given purine or pyrimidine base, which may in part explain the observationlg that some metal ions (e.g., Cu2+, Cd2+, Hg2+) lower the thermal denaturation temperature of DNA while others (e.g., Zn2+, Mn2+, Mg2+) have the reverse effect. Thus, for example, Zn2+ binds to N(7) in adeninelo while Cu2+ binds5 to N(9); moreover, Zn2+ binds to N(9) in guanine." Thus, these two metal ions would be expected to produce different effects on the hydrogen bonding scheme in a polynucleotide and, hence, a different degree of stabilization (or destabilization). The syntheses and structures of aza analogs of the nucleic acid constituents have also been widely studied during the past 2 or 3 y e a r ~ , ~ O mainly -~' as a result of the demonstrated but varying potency of some of these species (notably 8-azaguanine) as antineoplastic agent^.^^.^^ On the Journal of the American Chemical Society
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basis of structural models, it has been suggested that the biological activity of the ortho aza nucleosides (Le., 6-azapyrimidine or 8-azapurine nucleosides) may result from changes in their hydrogen bonding ability brought about by conformation changes.34 This postulate has recently received partial confirmation from theoretica13j and crystallographic studies, which have shown that the conformations of ortho aza nucleosides about the glycosyl bond are in a different angular range than are those of the naturally occurring n ~ c l e o s i d e s . ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ Since azapurines and azapyrimidines, therefore, are in common use in medical therapy, it is apparent that the interactions of naturally occurring metal ions with these bases are also of great importance. To our knowledge, however, no such studies have been reported to date, although we have noted in the 1iterature3l that HgC12 interacts sufficiently with 6-azauracil to allow the dissolution of this insoluble base in aqueous medium. Similarly, Zn2+ and Cu2+ form complexes with 6-azauracil and 6-azathymine, respectively, in aqueous solution,36but in alt of these cases it is the uncomplexed base which crystallizes from the solution. In order to overcome this lack of information, we have initiated a program of synthetic and structural chemistry to
April 30, 1975
